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不同激活方法对猪体外成熟卵母细胞孤雌发育的影响 总被引：10，自引：1，他引：9
研究了猪体外成熟卵母细胞的电激活、离子霉素激活和乙醇激活的方法。3种不同激活方法筛选试验表明,猪卵母细胞电激活最佳参数为电场强度130V/min,脉冲时程80μs的1次脉冲激活,即130V/mm-80μs-1次,其囊胚(发育)率为18.92%±8.48%(P>0.05);离子霉素激活的最佳条件为15μmol/L、激活时间40min,其囊胚率为21.27%±8.54%(P>0.05);乙醇激活最佳参数以9%乙醇激活处理3min,囊胚率为13.33%±7.64%。进一步对比试验表明,电激活和离子霉素激活处理的囊胚率和囊胚细胞数无显著差异(P>0.05),电激活的卵裂率明显高于乙醇激活(P<0.05),而囊胚率和细胞数差异不显著(P>0.05)。 相似文献
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[目的]了解玻璃化冷冻一解冻及不同孤雌激活条件对水牛卵母细胞发育潜能的影响,为构建完善的水牛卵母细胞孤雌激活培养体系奠定基础.[方法]利用玻璃化冷冻法对水牛体外成熟卵母细胞进行冷冻保存1～2d,解冻复苏后进行孤雌激活,以新鲜的体外成熟卵母细胞为对照,探讨离子霉素浓度、6-二甲氨基嘌呤(6-DMAP)浓度和处理时间对玻璃化冷冻复苏后水牛卵母细胞孤雌激活效果的影响.[结果]与新鲜的体外成熟卵母细胞相比,玻璃化冷冻—解冻复苏后水牛体外成熟卵母细胞孤雌激活的卵裂率、4-细胞率、8-细胞率和囊胚发育率均极显著降低(P＜0.01).水牛体外成熟卵母细胞经玻璃化冷冻—解冻复苏后,以3.5 μmol/L离子霉素激活5 min联合2mmol/L 6-DMAP培养2h的孤雌激活效果最佳,其卵裂率、4-细胞率、8-细胞率和囊胚发育率分别为60.6％、45.1％、33.7％和14.8％.[结论]水牛体外成熟卵母细胞经玻璃化冷冻—解冻复苏后,其激活阈值发生改变,因此不宜采用与新鲜体外成熟卵母细胞一致的激活程序进行孤雌激活. 相似文献
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Gerica LaVelle Betsy Cairo Jennifer P. Barfield 《Reproduction in domestic animals》2023,58(1):176-183
Pre-loading bovine sperm with cholesterol prior to freezing is known to increase cryosurvival, though the timing of capacitation in these sperm has not been evaluated. The objective of this study was to determine if there is a potential delay in capacitation timing in these sperm due to the increased cholesterol content. Flow cytometric evaluation was utilized to assess viability, and stain technology to assess acrosome intactness (Propidium Iodide/FITC-PNA), intracellular calcium levels (Propidium Iodide/FLUO 3-AM) and membrane fluidity (Merocyanine 540/YO-PRO-1). Cholesterol-loaded cyclodextrin (CLC) (2 mg/mL) improved post-thaw viability to 61% from 45% in control sperm (p < .05). The addition of ionomycin (0.05 mM) induced capacitation in sperm by 1 h, resulting in increased intracellular calcium and increased acrosome reaction, and consequently viability loss by 3 h. Treatment with CLC significantly decreased membrane fluidity in sperm (p < .05). In conclusion, CLC-treated sperm required 1 h more to capacitate when compared with non-treated sperm based on percentage of live cells with high membrane disorder (p < .05). Increased cryosurvival and viability over time was observed, but longer time to capacitate may hinder fertilization capacity and/or require adjustments to timing of in vitro fertilization. 相似文献
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The objective of the present study was to confirm previous results on the mediation of GnRH signal in tilapia by providing evidence from experiments in cultured pituitary cells and from perifusion experiments using a GnRH-antagonist. After 4 days in culture under identical conditions, cells taken from pituitaries of fish maintained at 26°C were more sensitive to GnRHa ([D-Ala6, Pro9-NEt]-LHRH) than those taken from fish maintained at 19°C. Cells from female pituitaries were more responsive than those from males. taGTH release in culture was augmented by Ca2+ ionophore (A23187; 1–100 μM) or ionomycin (0.02–10 μM). The response of perifused pituitary to GnRH was reduced by nimodipine (1–10 μM) indicating that Ca2+ influx via voltage-sensitive Ca2+ channels is involved in the stimulation of GTH release. Activation of protein kinase C by OAG (1-oleyl-2-acetyl glycerol; 0.16–160 μM) or TPA (1-O-tetra-decanoyl phorbol-13-acetate; 1.25–125 nM) resulted in a dose-dependent stimulation of taGTH release from cultured cells. Arachidonic acid (0.33–330 μM) also augmented the release of taGTH from the culture. Four sequential pulses of sGnRH (100 nM) at 2h intervals resulted in surges of taGTH release from perifused pituitary fragments; the surges were similar in magnitude with no signs of desensitization. Sequential stimulation with graded doses of sGnRH (0.1 nM to 1 μM) in the presence of GnRH-antagonist ([Pro2,6, Trp3]-GnRH) resulted in an attenuation of taGTH release. However, the GnRH-antagonist did not alter the pattern of forskolin-stimulated GTH release, indicating that forskolin stimulation is exerted at the level of the adenohypophyseal cells. It is concluded that, as in other vertebrates, the transduction of GnRH stimulation of GTH release involves Ca2+ influx through voltage-sensitive Ca2+ channels, mobilization of the ion from intracellular sources, arachidonic acid and activation of PKC. Adenylate cyclase-cAMP system us also involved in the mediation but its relationship with other transduction cascades requires further investigations. 相似文献
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对不同浓度离子霉素以及不同质量(A类或混合类)的猪卵丘细胞-卵母细胞复合体(COC s)对卵孤雌发育的效果影响进行了初步研究。结果表明:(1)A类猪COC s孤雌激活后的囊胚发育率极显著的高于混合类(30.11%vs 9.09%,P<0.01);(2)采用化学方法激活体外成熟后的猪去透明带卵母细胞,离子霉素浓度为5μm o l/L组激活后得到的卵裂率(80.35%),以及激活后48 h发育到4细胞阶段的孤雌胚胎率(62.50%)均极显著高于浓度2μm o l/L组(分别为62.22%和43.33%,P<0.01)。 相似文献
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主要比较了离子霉素、电脉冲及培养基更换对牛羊体外成熟卵母细胞孤雌激活的影响。两种方法对牛胚胎的研究中,牛胚胎卵裂率无显著差异(90.61%vs94.40%,P>0.05),而离子霉素激活胚胎的囊胚发育率极显著高于电激活方法(12.3%vs2.4%,P<0.01)。两种方法对羊胚胎的研究中,羊胚胎卵裂率无显著差异(72.4%vs77.4%,P>0.05)。但是离子霉素激活胚胎的囊胚发育率显著高于电激活方法(3.67%vs10.4%,P<0.05)。本试验还采用两种方法来培养牛化学激活孤雌胚。方法一是把化学激活牛体外成熟卵母细胞放于SOFaa(Synthetic Oviduct Fluid aa)培养基中连续培养7d;方法二是把化学激活牛体外成熟卵母细胞放于SOFaa培养基中4 d后,再转移进入含有10?S(Fetal Bovine Serum)的SOFaa培养基中继续培养3 d。试验结果表明:方法一中的囊胚发育率显著高于方法二(11.64%vs3.49%,P<0.01)。这也说明了更换培养基不利于其体外囊胚发育率。 相似文献
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牛体外成熟卵母细胞孤雌激活的研究 总被引：5，自引：0，他引：5
比较 3种化学激活方法和 4种电化学激活方法对牛体外成熟卵母细胞进行孤雌激活的效果。结果显示 ,单独用离子霉素和用离子霉素 + 6 DMAP +CB或 +CHX +CB激活牛卵母细胞 ,两者之间差异极显著 (P <0 0 1) ;用不同强度电脉冲 + 6 DMAP +CB激活牛卵母细胞 ,各组间卵裂率差异均不显著 (P >0 0 5 )。结果表明 ,虽然电化学激活方法最高可获得 82 9%的卵裂率 ,比化学激活方法中最好的高 4 8% ,但后者不需电融合仪 ,操作相对简便。 相似文献
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